The discovery and identification of diffusible factors that regulate skeletal morphogenesis have dramatically improved our understanding of the molecular events governing skeletal pattern formation. Genetic studies have confirmed the importance of these differentiation factors in the formation, growth and maintenance of the skeleton (Erlebacher et al., Cell, 80:371–378, 1995). Likewise, non-diffusible molecules, including components of the extracellular matrix and cell surface, are essential to patterning processes. One theory proposed for insect systems is that morphogenesis results from the (re)positioning of cells because of inherent characteristics such as differential adhesiveness (Nardi et al., J. Embryol. Exp. Morphol., 36:489–512, 1976). It is presently unknown whether analogous events occur in mammalian skeletal pattern formation.
In Drosophila melanogaster, the cuticle contains hairs and bristles arranged in a defined polarity, of which the pattern and orderly alignment reflect the polarity of the wing epidermis (Adler et al., Genetics, 126:401–416, 1990). Typically, these structures are aligned in parallel and point in the same direction as the body surface. Several genetic loci associated with epidermal cell polarity have been studied. One of the most thoroughly investigated is the frizzled (fz) locus. Frizzled encodes an integral membrane protein having seven potential transmembrane domains. The fz locus is required for cellular response to a tissue polarity signal as well as intercellular transmission of that signal along the proximal-distal wing axis (Vinson et al., Nature, 329:549–551, 1987; Vinson et al., Nature, 338:263–264, 1989). Mutations of the fz locus result in disruption of both cell-autonomous and noncell-autonomous functions of the fz gene. Strong fz mutations are associated with random orientation of wing hairs, while weaker mutations lead to hair and bristles randomly oriented parallel to neighboring cells with respect to the body axis (Vinson et al., Nature, 329:549–551, 1987). Frizzled also regulates mirror-symmetric pattern formation in the Drosophila eye (Zheng et al., Development, 121:3045–3055, 1995).
The rat and human homologs frizzled-1 and frizzled-2 (fz-1, fz-2) have been cloned and are expressed in a wide variety of tissues including kidney, liver, heart, uterus and ovary (Chan et al., J. Biol. Chem., 267:25202–25297, 1992; Zhao et al., Genomics, 27:373–373, 1995). Six novel mammalian frizzled homologs have now been identified (Wang et al., J. Biol. Chem., 271:4468–4476, 1996), each of which appears to be expressed in a distinct set of tissues during development or postnatally.
The basic form and pattern of the skeleton derived from lateral plate mesoderm are first recognizable when mesenchymal cells aggregate into regions of high cell density called condensations which subsequently differentiate into cartilage and bone, and continue to grow by cell proliferation, cell enlargement and matrix deposition. Published PCT Application No. WO 96/14335 discloses the isolation, cloning and in vivo chondrogenic activity of cartilage-derived morphogenetic proteins (CDMPs) which are members of the TGF-β superfamily. Genetic studies have demonstrated that disruption of condensations results in disturbed skeletal phenotypes (Erlebacher et la., Cell, 80:371–378, 1995). In humans, limb development takes place over a four week period from the fifth to the eighth week. The upper limbs develop slightly in advance of the lower limbs, although by the end of the period of limb development the two limbs are nearly synchronized. The most proximal parts of the limbs develop somewhat in advance of the more distal parts.
Recently, the number of secreted factors implicated in both limb and axial patterning has increased steadily (Sive, Genes Dev., 7:1–12, 1993; Dawid, J. Biol. Chem., 269:6259–6262, 1994; Hogan, Genes Dev., 10:1580–1594, 1996). Some of these factors are expressed in the Spemann organizer, the region of the Xenopus embryo implicated in specification of the dorsal axis and critical to dorso-ventral patterning of the vertebrate embryo. In contrast, the bone morphogenetic protein BMP-4 and Xwnt-8, a member of the Wnt family of growth factors, are expressed in presumptive ventral mesoderm and endoderm early in gastrulation, and are thought to act as positive ventral inducers (Hogan et al., supra; DeRobertis et al., Nature, 380:37–40, 1996; Christian et al., Genes Dev., 7:13–28, 1993). Several of these secreted factors are thought to produce their dorsalizing effects by binding to BMP-4 or a related TGF-β class signal and inactivating it. No secreted factor with Wnt binding activity has been identified.
Wnt proteins are implicated in a variety of developmental and neoplastic processes (Nusse et al., Cell, 69:1073–1087, 1992; Parr et al., Curr. Biol., 4:523–528, 1994; Moon, Bioessays, 15:91–97, 1993). The receptors for these proteins have not been identified. The Wnt family of proteins has been divided into two classes, I and II, based on their ability to induce axis duplication in Xenopus oocytes and their transforming activity in mammalian cells. Recently, Frizzled-class proteins were proposed as receptors for the Wnt growth factors (Wang et al., J. Biol. Chem., 271:4468–4476, 1996). This is supported by observations that Wingless protein (Wg), the Drosophila prototype of the Wnt family, binds to cells transfected with the frizzled2 gene (Dfz2). Moreover, addition of Wg to cells transfected with Dfz2 causes increased accumulation of Armadillo, a Drosophila homologue of β-catenin, an expected consequence of Wg signaling (Bhanot et al., Nature, 382:225–230, 1996). In Xenopus embryos, overexpression of rat frizzled-1 (Rfz-1) resulted in recruitment of Xwnt-8 and Xenopus dishevelled, a component of the Wnt signaling pathway, to the plasma membrane (Yang-Snyder et al., Current Biol, 6:1302–1306, 1996).
There are few known proteins which induce skeletal morphogenesis, as well as induction of nerve and muscle tissue growth. There are no known secreted proteins which will bind to and modulate the function of the Wnt proteins. Such proteins have tremendous therapeutic applications. The present invention provides such a multifaceted protein.